Acylated glucagon analogues

ABSTRACT

The invention provides materials and methods for the treatment of obesity and excess weight, diabetes, and other associated metabolic disorders. In particular, the invention provides novel acylated glucagon analogue peptides effective in such methods. The peptides may mediate their effect by having increased selectivity for the GLP-1 receptor as compared to human glucagon.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 12, 2019 isnamed “50412-087004 Sequence Listing” and is 46,885 bytes in size.

FIELD OF THE INVENTION

The present invention relates to acylated glucagon analogues and theirmedical use, for example in the treatment of obesity and excess weight,diabetes, and other metabolic disorders.

BACKGROUND OF THE INVENTION

Pre-proglucagon is a 158 amino acid precursor polypeptide that isdifferentially processed in the tissues to form a number of structurallyrelated proglucagon-derived peptides, including glucagon (Glu),glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), andoxyntomodulin (OXM). These molecules are involved in a wide variety ofphysiological functions, including glucose homeostasis, insulinsecretion, gastric emptying and intestinal growth, as well as regulationof food intake.

Glucagon is a 29-amino acid peptide that corresponds to amino acids 53to 81 of pre-proglucagon. Oxyntomodulin (OXM) is a 37 amino acid peptidewhich includes the complete 29 amino acid sequence of glucagon with anoctapeptide carboxyterminal extension (amino acids 82 to 89 ofpre-proglucagon, and termed “intervening peptide 1” or IP-1. The majorbiologically active fragment of GLP-1 is produced as a 30-amino acid,C-terminally amidated peptide that corresponds to amino acids 98 to 127of pre-proglucagon.

Glucagon helps maintain the level of glucose in the blood by binding toglucagon receptors on hepatocytes, causing the liver to releaseglucose—stored in the form of glycogen—through glycogenolysis. As thesestores become depleted, glucagon stimulates the liver to synthesizeadditional glucose by gluconeogenesis. This glucose is released into thebloodstream, preventing the development of hypoglycemia.

GLP-1 decreases elevated blood glucose levels by improvingglucose-stimulated insulin secretion and promotes weight loss chieflythrough decreasing food intake.

OXM is released into the blood in response to food ingestion and inproportion to meal calorie content. OXM has been shown to suppressappetite and inhibit food intake in humans (Cohen et al, Journal ofEndocrinology and Metabolism, 88, 4696-4701, 2003; WO 2003/022304). Inaddition to those anorectic effects, which are similar to those ofGLP-1, OXM must also affect body weight by another mechanism, since ratstreated with oxyntomodulin show less body weight gain than pair-fed rats(Bloom, Endocrinology 2004, 145, 2687). Treatment of obese rodents withOXM also improves their glucose tolerance (Parlevliet et al, Am JPhysiol Endocrinol Metab, 294, E142-7, 2008) and suppresses body weightgain (WO 2003/022304).

OXM activates both the glucagon and the GLP-1 receptors with a two-foldhigher potency for the glucagon receptor over the GLP-1 receptor, but isless potent than native glucagon and GLP-1 on their respectivereceptors. Human glucagon is also capable of activating both receptors,though with a strong preference for the glucagon receptor over the GLP-1receptor. GLP-1 on the other hand is not capable of activating glucagonreceptors. The mechanism of action of oxyntomodulin is not wellunderstood. In particular, it is not known whether some of theextrahepatic effects of the hormone are mediated through the GLP-1 andglucagon receptors, or through one or more unidentified receptors.

Other peptides have been shown to bind and activate both the glucagonand the GLP-1 receptor (Hjort et al, Journal of Biological Chemistry,269, 30121-30124, 1994) and to suppress body weight gain and reduce foodintake (see, for example, WO 2006/134340, WO 2007/100535, WO 2008/10101,WO 2008/152403, WO 2009/155257, WO 2009/155258, WO2010/070252,WO2010/070253, WO2010/070255, WO2010/070251, WO2011/006497,WO2011/160630, WO2011/160633, WO2013/092703, WO2014/041195.

Obesity is a globally increasing health problem associated with variousdiseases, particularly cardiovascular disease (CVD), type 2 diabetes,obstructive sleep apnea, certain types of cancer, and osteoarthritis. Asa result, obesity has been found to reduce life expectancy. According to2005 projections by the World Health Organization there are 400 millionadults (age >15) classified as obese worldwide. In the US, obesity isnow believed to be the second-leading cause of preventable death aftersmoking.

The rise in obesity drives an increase in diabetes, and approximately90% of people with type 2 diabetes may be classified as obese. There are246 million people worldwide with diabetes, and by 2025 it is estimatedthat 380 million will have diabetes. Many have additional cardiovascularrisk factors, including high/aberrant LDL and triglycerides and low HDL.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a compound having the formula:R¹—P¹—P²—R²

wherein

R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

R² is OH or NH₂;

P¹ is a peptide having the sequence:

(SEQ ID NO: 65) H-X2-X3-GTFTSDYSKYLDSΨAAHDFVEWLLSA

wherein:

X2 is selected from Aib, Ala, D-Ala, Ser, N-Me-Ser, Ac3c, Ac4c and Ac5c;

X3 is selected from Gln and His;

P² is absent or is a sequence of 1-20 amino acid units independentlyselected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu,Lys, Arg, Dbu, Dpr and Orn;

or a pharmaceutically acceptable salt or solvate thereof;

Ψ is a residue of Lys, Arg, Orn or Cys in which the side chain isconjugated to a substituent having the formula —Z²—Z¹;

—Z¹ is a fatty chain having a polar group at one end of the chain and aconnection to Z², —X— at the end of the chain distal from the polargroup,

wherein the polar group comprises a carboxylic acid or a carboxylic acidbioisostere, a phosphonic acid, or a sulfonic acid group;

and —X— is a bond, —CO—, —SO—, or —SO₂—;

—Z²— is a spacer of formula:

wherein:

each Y is independently —NH, —NR, —S or —O, where R is alkyl, aprotecting group or forms a linkage to another part of the spacer Z²;

each X is independently a bond, CO—, SO—, or SO₂—;

with the proviso that when Y is —S, X is a bond;

each V is independently a bivalent organic moiety linking Y and X;

and n is 1-10;

or a pharmaceutically acceptable salt or solvate thereof.

P¹ may have the sequence:

(SEQ ID NO: 66) H-Aib-QGTFTSDYSKYLDSΨAAHDFVEWLLSA   e.g. (SEQ ID NO: 67)H-Aib-QGTFTSDYSKYLDS-K([15-carboxy- pentadecanoyl]-isoGlu)-AAHDFVEWLLSA.

The compound of the invention may be:

(SEQ ID NO: 68) H-H-Aib-QGTFTSDYSKYLDSΨAAHDFVEWLLSA-NH₂ e.g.(SEQ ID NO: 69) H-H-Aib-QGTFTSDYSKYLDS-K([15-carboxy-pentadecanoyl]-isoGlu)-AAHDFVEWLLSA-NH₂.

In a second aspect, the invention provides a compound having theformula:R¹—P¹—P²—R²

wherein

R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

R² is OH or NH₂;

P¹ is a peptide having the sequence:

(SEQ ID No: 1) His-X2-X3-GTFTSDYSKYL-X15-X16-X17-X18-A-X20-DFI-X24-WLE-X28-A

wherein:

X2 is selected from Aib, Ac3c, Ac4c and Ac5c;

X3 is selected from Gln and His;

X15 is selected from Asp and Glu;

X16 is selected from Glu and Ψ;

X17 is selected from Arg and Ψ;

X18 is selected from Ala and Arg;

X20 is selected from Lys and His;

X24 is selected from Glu and Ψ;

X28 is selected from Ser and Ψ;

and P² is absent or is a sequence of 1-20 amino acid units independentlyselected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu,Lys, Arg, Dbu, Dpr and Orn;

wherein the compound contains one and only one Ψ

and wherein said Ψ is a residue of Lys, Arg, Orn or Cys in which theside chain is conjugated to a substituent having the formula —Z²—Z¹;

—Z¹ is a fatty chain having a polar group at one end of the chain and aconnection to Z², —X— at the end of the chain distal from the polargroup,

wherein the polar group comprises a carboxylic acid or a carboxylic acidbioisostere, a phosphonic acid, or a sulfonic acid group;

and —X— is a bond, —CO—, —SO—, or —SO₂—;

—Z²— is a spacer of formula:

wherein:

each Y is independently —NH, —NR, —S or —O, where R is alkyl, aprotecting group or forms a linkage to another part of the spacer Z²;

each X is independently a bond, CO—, SO—, or SO₂—;

with the proviso that when Y is —S, X is a bond;

each V is independently a bivalent organic moiety linking Y and X;

and n is 1-10;

or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments of the second aspect:

X2 is selected from Aib and Ac4c;

X3 is Gln;

X15 is selected from Asp and Glu;

X16 is Ψ;

X17 is Arg;

X18 is Ala;

X20 is selected from Lys and His;

X24 is Glu;

X28 is Ser.

Useful combinations of residues include the following:

X2 is Ac4c and X20 is Lys;

X2 is Aib and X20 is His.

Additionally or alternatively, it may be desirable that X2 is Aib if X15is E

or that X15 is D if X2 is Ac4c.

Particularly interesting substituents Z²Z¹ include[17-carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3 and[17-carboxy-heptadecanoyl]-isoGlu-GSGSGG (SEQ ID NO: 34).

P¹ may have a sequence selected from:

(SEQ ID NO: 4) H-Aib-QGTFTSDYSKYLDΨRAAKDFIEWLESA; (SEQ ID NO: 5)H-Aib-QGTFTSDYSKYLDΨRAAKDFIEWLESA; (SEQ ID NO: 6)H-Aib-QGTFTSDYSKYLEΨRAAKDFIEWLESA; (SEQ ID NO: 7)H-Ac4c-QGTFTSDYSKYLDΨRAAKDFIEWLESA; and (SEQ ID NO: 8)H-Aib-QGTFTSDYSKYLEΨRAAHDFIEWLESA, e.g. from (SEQ ID NO: 35)H-Aib-QGTFTSDYSKYLD-K([17-carboxy- heptadecanoyl]-isoGlu-Peg3-Peg3)-RAAKDFIEWLESA; (SEQ ID NO: 36) H-Aib-QGTFTSDYSKYLD-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)- RAAKDFIEWLESA; (SEQ ID NO: 37)H-Aib-QGTFTSDYSKYLE-K([17-carboxy- heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA; (SEQ ID NO: 38) H-Ac4c-QGTFTSDYSKYLD-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)- RAAKDFIEWLESA;  and (SEQ ID NO: 39)H-Aib-QGTFTSDYSKYLE-K([17-carboxy- heptadecanoyl]-isoGlu-Peg3-Peg3)-RAAHDFIEWLESA.

The compound of the invention may be selected from:

(SEQ ID NO: 9) H-H-Aib-QGTFTSDYSKYLDΨRAAKDFIEWLESA-NH₂; (SEQ ID NO: 10)H-H-Aib-QGTFTSDYSKYLDΨRAAKDFIEWLESA-NH₂; (SEQ ID NO: 11)H-H-Aib-QGTFTSDYSKYLEΨRAAKDFIEWLESA-NH₂; (SEQ ID NO: 12)H-H-Ac4c-QGTFTSDYSKYLDΨRAAKDFIEWLESA-NH₂;  and (SEQ ID NO: 13)H-H-Aib-QGTFTSDYSKYLEΨLRAAHDFIEWLESA-NH₂, e.g. from (SEQ ID NO: 40)H-H-Aib-QGTFTSDYSKYLD-K([17-carboxy- heptadecanoyl]-isoGlu-Peg3-Peg3)-RAAKDFIEWLESA-NH₂; (SEQ ID NO: 41) H-H-Aib-QGTFTSDYSKYLD-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)- RAAKDFIEWLESA-NH₂; (SEQ ID NO: 42)H-H-Aib-QGTFTSDYSKYLE-K [(17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH₂; (SEQ ID NO: 43)H-H-Ac4c-QGTFTSDYSKYLD-K([17-carboxy- heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH₂;  and (SEQ ID NO: 44)H-H-Aib-QGTFTSDYSKYLE-K([17-carboxy- heptadecanoyl]-isoGlu-Peg3-Peg3)-RAAHDFIEWLESA-NH₂.

In alternative embodiments of the second aspect:

X2 is selected from Aib and Ac4c;

X3 is selected from Gln and His;

X15 is Asp;

X16 is Glu;

X17 is selected from Arg and Ψ;

X18 is selected from Ala and Arg;

X20 is Lys;

X24 is selected from Glu and Ψ;

X28 is selected from Ser and Ψ;

In some embodiments, when X28 is Ψ, X2 is Ac4c.

In some embodiments, when X3 is His, X2 is Ac4c and X17 is Ψ.

In some embodiments, when X17 is Ψ, Z²Z¹ is[17-carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3 or[17-carboxy-heptadecanoyl]-isoGlu.

In some embodiments, when X24 or X28 is Ψ, Z²Z¹ is[17-carboxy-heptadecanoyl]-isoGlu-GSGSGG (SEQ ID NO: 34).

P¹ may have a sequence selected from:

(SEQ ID NO: 14) H-Aib-QGTFTSDYSKYLDEΨAAKDFIEWLESA; (SEQ ID NO: 15)H-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA; (SEQ ID NO: 16)H-Ac4c-HGTFTSDYSKYLDEΨRAKDFIEWLESA; (SEQ ID NO: 17)H-Ac4c-QGTFTSDYSKYLDEΨAAKDFIEWLESA;  (SEQ ID NO: 18)H-Ac4c-QGTFTSDYSKYLDEΨRAKDFIEWLESA;  (SEQ ID NO: 19)H-Aib-QGTFTSDYSKYLDERAAKDFIΨWLESA;  (SEQ ID NO: 20)H-Ac4c-QGTFTSDYSKYLDERAAKDFIΨWLESA; (SEQ ID NO: 21)H-Ac4c-QGTFTSDYSKYLDERRAKDFIΨWLESA;  (SEQ ID NO: 22)H-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLEΨA;  and  (SEQ ID NO: 23)H-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLEΨA,  e.g. from (SEQ ID NO: 45)H-Aib-QGTFTSDYSKYLDE-K([17-carboxy- heptadecanoyl]-isoGlu)-AAKDFIEWLESA; (SEQ ID NO: 46)H-Ac4c-QGTFTSDYSKYLDE-K([17-carboxy- heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA; (SEQ ID NO: 47) H-Ac4c-HGTFTSDYSKYLDE-K([17-carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)- RAKDFIEWLESA; (SEQ ID NO: 48)H-Ac4c-QGTFTSDYSKYLDE-K([17-carboxy-heptadecanoyl]-isoGlu)-AAKDFIEWLESA;  (SEQ ID NO: 49)H-Ac4c-QGTFTSDYSKYLDE-K([17-carboxy-heptadecanoyl]-isoGlu)-RAKDFIEWLESA; (SEQ ID NO: 50)H-Aib-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA; (SEQ ID NO: 51)H-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA; (SEQ ID NO: 52)H-Ac4c-QGTFTSDYSKYLDERRAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA; (SEQ ID NO: 53)H-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A; and (SEQ ID NO: 54)H-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A.

The compound of the invention may be selected from:

 (SEQ ID NO: 24) H-H-Aib-QGTFTSDYSKYLDE1PAAKDFIEWLESA-NH₂;(SEQ ID NO: 25) H-H-Ac4c-QGTFTSDYSKYLDELPRAKDFIEWLESA-NH₂;(SEQ ID NO: 26) H-H-Ac4c-HGTFTSDYSKYLDELPRAKDFIEWLESA-NH₂;(SEQ ID NO: 27) H-H-Ac4c-QGTFTSDYSKYLDELPAAKDFIEWLESA-NH₂; (SEQ ID NO: 28) H-H-Ac4c-QGTFTSDYSKYLDELPRAKDFIEWLESA-NH₂;(SEQ ID NO: 29) H-H-Aib-QGTFTSDYSKYLDERAAKDFILPWLESA-NH₂;(SEQ ID NO: 30) H-H-Ac4c-QGTFTSDYSKYLDERAAKDFILPWLESA-NH₂; (SEQ ID NO: 31) H-H-Ac4c-QGTFTSDYSKYLDERRAKDFILPWLESA-NH₂;  (SEQ ID NO: 32) H-H-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLELPA-NH₂; and(SEQ ID NO: 33) H-H-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLELPA-NH₂, e.g. from(SEQ ID NO: 55) H-H-Aib-QGTFTSDYSKYLDE-K([17-carboxy-heptadecanoyq-isoGlu)-AAKDFIEWLESA-NH₂; (SEQ ID NO: 56)H-H-Ac4c-QGTFTSDYSKYLDE-K([17-carboxy- heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA-NH₂; (SEQ ID NO: 57) H-H-Ac4c-HGTFTSDYSKYLDE-K([17-carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)- RAKDFIEWLESA-NH₂; (SEQ ID NO: 58)H-H-Ac4c-QGTFTSDYSKYLDE-K([17-carboxy-heptadecanoyl]-isoGlu)-AAKDFIEWLESA-NH₂; (SEQ ID NO: 59)H-H-Ac4c-QGTFTSDYSKYLDE-K([17-carboxy-heptadecanoyl]-isoGlu)-RAKDFIEWLESA-NH₂; (SEQ ID NO: 60)H-H-Aib-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH₂; (SEQ ID NO: 61)H-H-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH₂; (SEQ ID NO: 62)H-H-Ac4c-QGTFTSDYSKYLDERRAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH₂; (SEQ ID NO: 63)H-H-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH₂; and (SEQ ID NO: 64)H-H-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-A-NH₂.

For the avoidance of doubt, in all aspects of the invention, thosepositions which are not expressly stated to permit variability are fixedand thus may only include the stated residue.

In all aspects, the compound of the invention comprises a residue Ψ,i.e. a residue selected from Lys, Arg, Orn and Cys in which the sidechain is conjugated to a substituent —Z²—Z¹— as described in more detailbelow.

The substituent is conjugated to the functional group at the distal endof the side chain from the alpha-carbon. The normal ability of the Lys,Arg, Orn or Cys side chain to participate in interactions mediated bythat functional group (e.g. intra- and inter-molecular interactions) maytherefore be reduced or completely eliminated by the presence of thesubstituent. Thus, the overall properties of the compound may berelatively insensitive to changes in the actual amino acid present asresidue Ψ. Consequently, it is believed that any of the residues Lys,Arg, Orn and Cys may be present at any position where Ψ is permitted.However, in certain embodiments, it may be advantageous that the aminoacid component of Ψ is Lys.

In some embodiments, —Z¹ is an acyl group of formula:A-B-Alk-(CO)—

or a sulfonyl group of formula:A-B-Alk-(SO₂)—;

A is —COOH or a carboxylic acid bioisostere;

B is a bond, C₆arylene, or C₆arylene-O—;

Alk is a saturated or unsaturated fatty chain of 6 to 18 carbon atoms inlength, optionally substituted with one or more substituents selectedfrom fluoro, trifluoromethyl, hydroxymethyl, amino, hydroxyl,C₁₋₄alkoxy, oxo, and carboxyl;

—Z²— is —S_(A)—, —S_(A)—S_(B)—, or —S_(B)—S_(A)—;

—S_(A)— is a single amino acid residue selected from γ-Glu, α-Glu,α-Asp, β-Asp, Ala, β-Ala (3-aminopropanoic acid), and Gaba(4-aminobutanoic acid);

—S_(B)— is a linker of general formula:

wherein n is 1-10 and each P_(U) is independently selected from P_(U)^(i) and P_(U) ^(iii);

each P_(U) ^(i) is independently a natural or unnatural amino acidresidue; and

each P_(U) ^(iii) is independently a residue of general formula:

wherein m is 0-5 and p is 1, 3, 4, or 5.

In any aspect of the invention, R¹ may be selected from H and C₁₋₄ alkyl(e.g. methyl).

The compounds of the invention are glucagon analogue peptides.References herein to a glucagon analogue peptide should be construed asreferences to a compound of the invention or to a peptide P¹ or P¹-P² asthe context requires. Reference to a compound of the invention should betaken to include any pharmaceutically acceptable salt (e.g. an acetateor chloride salt) or solvate thereof, unless otherwise stated orexcluded by context.

The invention provides a composition comprising a compound of theinvention as defined herein (including pharmaceutically acceptable saltsor solvates thereof, as already described) in admixture with a carrier.In preferred embodiments, the composition is a pharmaceuticalcomposition and the carrier is a pharmaceutically acceptable carrier.The glucagon analogue peptide may be in the form of a pharmaceuticallyacceptable salt of the glucagon analogue.

The compounds described herein find use, inter alia, in preventingweight gain or promoting weight loss. By “preventing” is meantinhibiting or reducing when compared to the absence of treatment, and isnot necessarily meant to imply complete cessation of weight gain. Thepeptides may cause a decrease in food intake and/or increased energyexpenditure, resulting in the observed effect on body weight.Independently of their effect on body weight, the compounds of theinvention may have a beneficial effect on glucose control and/or oncirculating cholesterol levels, being capable of lowering circulatingLDL levels and increasing HDL/LDL ratio. Thus the compounds of theinvention can be used for direct or indirect therapy of any conditioncaused or characterised by excess body weight, such as the treatmentand/or prevention of obesity, morbid obesity, obesity linkedinflammation, obesity linked gallbladder disease, obesity induced sleepapnea. They may also be used for the prevention of conditions caused orcharacterised by inadequate glucose control or dyslipidaemia (e.g.elevated LDL levels or reduced HDL/LDL ratio), diabetes (especially Type2 diabetes), metabolic syndrome, hypertension, atherogenic dyslipidemia,atherosclerosis, arteriosclerosis, coronary heart disease, peripheralartery disease, stroke or microvascular disease. Their effects in theseconditions may be as a result of or associated with their effect on bodyweight, or may be independent thereof.

The invention also provides a compound of the invention for use in amethod of medical treatment, particularly for use in a method oftreatment of a condition as described above.

The invention also provides the use of a compound of the invention inthe preparation of a medicament for the treatment of a condition asdescribed above.

The compound of the invention may be administered as part of acombination therapy with an agent for treatment of diabetes, obesity,dyslipidaemia or hypertension.

In such cases, the two active agents may be given together orseparately, and as part of the same pharmaceutical formulation or asseparate formulations.

Thus the compound of the invention can be used in combination with ananti-diabetic agent including but not limited to a biguanide (e.g.metformin), a sulfonylurea, a meglitinide or glinide (e.g. nateglinide),a DPP-IV inhibitor, an SGLT2 inhibitor, a glitazone, an insulin, or aninsulin analogue. Examples of insulin analogues include but are notlimited to Lantus™, Novorapid™, Humalog™, Novomix™, Actraphane HM™,Levemir™ and Apidra™.

The compound can further be used in combination with an anti-obesityagent including but not limited to a glucagon-like peptide receptor 1agonist, peptide YY or analogue thereof, cannabinoid receptor 1antagonist, lipase inhibitor, melanocortin receptor 4 agonist, melaninconcentrating hormone receptor 1 antagonist, phentermine (alone or incombination with topiramate), a combination of norepinephrine/dopaminereuptake inhibitor and opioid receptor antagonist (e.g. a combination ofbupropion and naltrexone), or a serotonergic agent (e.g. lorcaserin).

The compound can further be used in combination with ananti-hypertension agent including but not limited to anangiotensin-converting enzyme inhibitor, angiotensin II receptorblocker, diuretic, beta-blocker, or calcium channel blocker.

The compound can be used in combination with an anti-dyslipidaemia agentincluding but not limited to a statin, a fibrate, a niacin or acholesterol absorption inhibitor.

Thus the invention further provides a composition or therapeutic kitcomprising a compound of the invention and for example an anti-diabeticagent, anti-obesity agent, anti-hypertension agent or anti-dyslipidaemiaagent as described above. Also provided is such a composition ortherapeutic kit for use in a method of medical treatment, especially fortreatment of a condition as described above.

The compound of the invention may be made by synthetic chemistry.Accordingly the invention provides a method of synthesis of a compoundof the invention.

The invention may also be made by a combination of recombinant andsynthetic methods. The method may comprise expressing a precursorpeptide sequence, optionally purifying the compound thus produced, andadding or modifying one or more amino acids to produce a compound of theinvention or a compound comprising the amino acid sequence P¹ or P¹-P².The step of modification may comprise introduction of an Orn residue(e.g. by modification of a precursor residue) and/or introduction of asubstituent Z2Z1 at the site of a residue Ψ.

The precursor peptide may be expressed from a nucleic acid encoding theprecursor peptide in a cell or a cell-free expression system comprisingsuch a nucleic acid.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this specification, the conventional one letter and threeletter codes for naturally occurring amino acids are used, as well asgenerally accepted abbreviations for other amino acids, such as Aib(α-aminoisobutyric acid), Orn (ornithine), Dbu (2,4-diaminobutyricacid), Dpr (2,3-diaminopropanoic acid), Ac3c(1-amino-cyclopropanecarboxylic acid), Ac4c(1-amino-cyclobutanecarboxylic acid) and Ac5c(1-amino-cyclopentanecarboxylic acid).

Ac3c, Ac4c and Ac5c have similar structures and are to some extentinterchangeable, although Ac4c may be preferred.

Glucagon is a 29-amino acid peptide that corresponds to amino acids 53to 81 of pre-proglucagon and has the sequenceHis-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr.Oxyntomodulin (OXM) is a 37 amino acid peptide which includes thecomplete 29 amino acid sequence of glucagon with an octapeptidecarboxyterminal extension (amino acids 82 to 89 of pre-proglucagon,having the sequence Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala and termed“intervening peptide 1” or IP-1; the full sequence of humanoxyntomodulin is thusHis-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala).The major biologically active fragment of GLP-1 is produced as a30-amino acid, C-terminally amidated peptide that corresponds to aminoacids 98 to 127 of pre-proglucagon.

The term “native glucagon” thus refers to native human glucagon havingthe sequenceH-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-OH.

Amino acids within the sequence P¹ of the compounds of the invention canbe considered to be numbered consecutively from 1 to 29 in theconventional N-terminal to C-terminal direction. Reference to a“position” within P¹ should be construed accordingly, as shouldreference to positions within native human glucagon and other molecules.

A compound of the invention may comprise a C-terminal peptide sequenceP² of 1-20 amino acids, for example to stabilise the conformation and/orsecondary structure of the glucagon analogue peptide, and/or to renderthe glucagon analogue peptide more resistant to enzymatic hydrolysis,e.g. as described in WO99/46283.

When present, P² represents a peptide sequence of 1-20 amino acidresidues, e.g. in the range of 1-15, more preferably in the range of1-10, in particular in the range of 1-7 amino acid residues, e.g., 1, 2,3, 4, 5, 6 or 7 amino acid residues, such as 6 amino acid residues. Eachof the amino acid residues in the peptide sequence P² may independentlybe selected from Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu(2,4-diaminobutyric acid), Dpr (2,3-diaminopropanoic acid) and Orn(ornithine). Preferably, the amino acid residues are selected from Ser,Thr, Tyr, Glu, Lys, Arg, Dbu, Dpr and Orn, more preferably selectedexclusively from Glu, Lys, and Cys. The above-mentioned amino acids mayhave either D- or L-configuration, which in certain embodiments, have anL-configuration. Particularly preferred sequences P² are sequences offour, five, six or seven consecutive lysine residues (i.e. Lys₃, Lys₄,Lys₅, Lys₆ or Lys₇), and particularly five or six consecutive lysineresidues. Other exemplary sequences of P² are shown in WO 01/04156.Alternatively the C-terminal residue of the sequence P² may be a Cysresidue. This may assist in modification (e.g. PEGylation, orconjugation to albumin) of the compound. In such embodiments, thesequence P² may, for example, be only one amino acid in length (i.e.P²=Cys) or may be two, three, four, five, six or even more amino acidsin length. The other amino acids therefore serve as a spacer between thepeptide P¹ and the terminal Cys residue.

The peptide sequence P² has no more than 25% sequence identity with thecorresponding sequence of the IP-1 portion of human OXM (which has thesequence Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala).

“Percent (%) amino acid sequence identity” of a given peptide orpolypeptide sequence with respect to another polypeptide sequence (e.g.IP-1) is calculated as the percentage of amino acid residues in thegiven peptide sequence that are identical with correspondinglypositioned amino acid residues in the corresponding sequence of thatother polypeptide when the two are aligned with one another, introducinggaps for optimal alignment if necessary. % identity values may bedetermined using WU-BLAST-2 (Altschul et al., Methods in Enzymology,266:460-480 (1996)). WU-BLAST-2 uses several search parameters, most ofwhich are set to the default values. The adjustable parameters are setwith the following values: overlap span=1, overlap fraction=0.125, wordthreshold (T)=11. A % amino acid sequence identity value is determinedby the number of matching identical residues as determined byWU-BLAST-2, divided by the total number of residues of the referencesequence (gaps introduced by WU-BLAST-2 into the reference sequence tomaximize the alignment score being ignored), multiplied by 100.

Thus, when P² is aligned optimally with the 8 amino acids of IP-1, ithas no more than two amino acids which are identical with thecorresponding amino acids of IP-1.

In certain embodiments, P² is absent.

Ψ is a residue of Lys, Arg, Orn or Cys whose side chain is conjugated toa substituent Z²—Z¹. Without wishing to be bound by any particulartheory, it is thought that the substituent binds plasma proteins (e.g.albumin) in the blood stream, thus shielding the compounds of theinvention from enzymatic degradation and thereby enhancing the half-lifeof the compounds. It may also modulate the potency of the compound, e.g.with respect to the glucagon receptor and/or the GLP-1 receptor.

The Group Z¹

Z¹ is a fatty chain having a connection to Z², referred to herein as —X—and, at the end of the chain distal from the connection to Z², a polargroup. —X— may be, for example, a bond, acyl (—CO—), sulfinyl (—SO—), orsulfonyl (—SO₂—), the connection being located at the ω-position withrespect to the polar group, that is, at the end of the chain distal fromthe polar group.

Preferably, the polar group is an acidic or weakly acid group, forexample a carboxylic acid or a carboxylic acid bioisostere, aphosphonate, or a sulfonate. The polar group may have a pK_(a) ofbetween −2 and 12 in water, more preferably between 1 and 7, morepreferably between 3 and 6. Certain preferred polar groups have a pK_(a)of between 4 and 5.

The polar group preferably comprises a carboxylic acid or carboxylicacid bioisostere. Suitable carboxylic acid bioisosteres are known in theart. Preferably the bioisostere has a proton having a pK_(a) similar tothe corresponding carboxylic acid. Examples of suitable bioisoteres mayinclude, not by way of limitation, tetrazole, acylsulfomides,acylhydroxylamine, and squaric acid derivatives, as shown below (---indicates the point of attachment):

The polar group may be a group of formula A-B—, wherein A is acarboxylic acid (—COOH) or a carboxylic acid bioisostere, a phosphonicacid (—P(O)(OH)₂), or a sulfonic acid (—SO₂OH) group, and B is a bond orlinker between A and the fatty chain. In some embodiments, the polargroup is —COOH, that is, A is —COOH and B is a bond.

When B is a linker, it may be a cycloalkylene, heterocycloalkylene,C₆arylene, or C₅₋₆heteroarylene, or C₆arylene-O— orC₅₋₆heteroarylene-O—.

When B is phenylene it may, for example, be selected from 1,2-phenylene,1,3-phenylene, 1,4-phenylene, preferably 1,4-phenylene (so that A-B— isa 4-benzoic acid substituent or 4-benzoic acid bioisostere). When B isphenylene-O—, it may, for example, be selected from 1,2-phenylene-O—,1,3-phenylene-O—, 1,4-phenylene-O—, preferably 1,4-phenylene-O. Eachphenylene of B may be optionally substituted with one or moresubstituents selected from fluoro, methyl, trifluoromethyl, amino,hydroxyl, and C₁₋₄alkoxy, preferably methoxy. It will be appreciatedthat substituent identity and position may be selected to subtly alterthe pK_(a) of the polar group. Suitable inductively or mesomericallyelectron-withdrawing or donating groups and their positional effects areknown in the art. In some embodiments, B may be C₅₋₆heteroarylene, forexample, pyridinylene or thiofuranylene, and may be optionallysubstituted as described.

For example, in some embodiments, A-B— may be selected from:

Preferably, A is —COOH. In some preferred polar groups, A is acarboxylic acid and B is C₆arylene-O—.

Fatty chain as used herein refers to a moiety comprising a chain ofcarbon atoms, the carbon atoms being predominantly substituted withhydrogen or hydrogen-like atoms, for example, a hydrocarbon chain. Suchfatty chains are often referred to as lipophilic, although it will beappreciated that substitution may alter the lipophilic properties of theoverall molecule.

The fatty chain may by aliphatic. It may be entirely saturated or mayinclude one or more double or triple bonds. Each double bond, ifpresent, may be in the E or Z configuration. The fatty chain may alsohave one or more cycloalkylene or heterocycloalkylene moieties in itslength, and additionally or alternatively may have one or more aryleneor heteroarylene moieties in its length. For example, the fatty chainmay incorporate a phenylene or piperazinylene moiety in its length as,for example, shown below (wherein --- represents the points ofattachment within the chain).

The fatty chain may be derived from a fatty acid, for example, it may bederived from a medium-chain fatty acid (MCFA) with an aliphatic tail of6-12 carbon atoms, a long-chain fatty acid (LCFA) with an aliphatic tailof 13-21 carbon atoms, or a very long-chain fatty acid (LCFA) with analiphatic tail of 22 carbon atoms or more. Examples of linear saturatedfatty acids from which suitable fatty chains may be derived includetridecylic (tridecanoic) acid, myristic (tetradecanoic) acid,pentadecylic (pentadecanoic) acid, palmitic (hexadecanoic) acid, andmargaric (heptadecanoic) acid. Examples of linear unsaturated fattyacids from which suitable fatty chains may be derived includemyristoleic acid, palmitoleic acid, sapienic acid and oleic acid.

The fatty chain may be connected to Z² by an amide linkage, asulfinamide linkage, a sulfonamide linkage, or by an ester linkage, orby an ether, thioether or amine linkage. Accordingly, the fatty chainmay have at the ω position, that is, the position distal to the polargroup, a bond to Z² or an acyl (—CO—), sulfinyl (—SO—), or sulfonyl(—SO₂—) group. Preferably, the fatty chain has an acyl (—CO—) group atthe position distal to the polar group and is connected to Z² by anamide or ester linkage.

In some embodiments, Z¹ is a group of formula:A-B-Alk-X—

where A-B— is the polar group defined above, X is a bond, acyl (—CO—),sulfinyl (—SO—), or sulfonyl (—SO₂—), and Alk is a fatty chain that maybe optionally substituted with one or more substituents. The fatty chainis preferably 6 to 18 carbon atoms in length (e.g. a C₆₋₁₈alkylene),more preferably, 8 to 18 carbons in length (e.g. a C₈₋₁₈alkylene), morepreferably, 12 to 16 carbons in length (e.g. C₁₂₋₁₆alkylene), and may besaturated or unsaturated. Preferably, Alk is saturated, that is,preferably Alk is alkylene.

In some embodiments, Z¹ is an acyl group of formula:A-B-Alk-(CO)—

or a sulfonyl group of formula:A-B-Alk-(SO₂)—.

Optional substituents on the fatty chain may be independently selectedfrom fluoro, C₁₋₄alkyl, preferably methyl; trifluoromethyl,hydroxymethyl, amino, hydroxyl, C₁₋₄ alkoxy, preferably methoxy; oxo,and carboxyl, and may be independently located at any point along thechain. In some embodiments, each optional substituent is selected fromfluoro, methyl, and hydroxyl. Where more than one substituent ispresent, substituents may be the same or different. Preferably, thenumber of substituents is 0 to 3; more preferably the fatty chain isunsubstituted.

Preferably, Z¹ is an acyl group of formula:A-B-alkylene-(CO)—

Where A and B are as defined above.

In some embodiments, Z¹ is:

4-carboxyphenoxynonanoyl HOOC—C₆H₄—O—(CH₂)₈—(CO)—.

Certain preferred Z¹ are derived from long-chain saturatedα,ω-dicarboxylic acids of formula HOOC—(CH₂)₁₂₋₁₈—COOH, preferably,long-chain saturated α,ω-dicarboxylic acids having an even number ofcarbon atoms in the aliphatic chain. For example, and not by way oflimitation, Z¹ may be:

13-carboxytridecanoyl HOOC—(CH₂)₁₂—(CO)—

15-carboxpentadecanoyl HOOC—(CH₂)₁₄—(CO)—; or

17-carboxyheptadecanoyl HOOC—(CH₂)₁₆—(CO)—.

The carboxylic acid group may be replaced by a bioisostere as detailedherein.

The Group Z²

Z² is spacer that connects Z¹ to the side chain of the amino acidcomponent of Ψ. At its most general, Z² is a spacer bound at oneterminus by Y, which may be a nitrogen, oxygen or sulfur atom, and atthe other terminus by X, which may be a bond or an acyl (—CO—), sulfinyl(—SO—), or sulfonyl (—SO₂—). Accordingly, Z² may be a spacer of formula(--- indicate points of attachment):

wherein:

Y may be —NH, —NR, —S or —O₂—, where R may be alkyl, a protecting groupor may form a linkage to another part of the spacer, with the remainingvalency forming a linkage to Z¹;

X may be a bond, CO—, SO—, or SO₂—, with the remaining valency forming alinkage to the side chain of the amino acid component of Ψ;

V is a bivalent organic moiety linking Y and X;

and n may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Where n is 2 or more, eachY, V, and X is independent of every other Y, V, and X.

Accordingly, Z² may be bound at each side by amide, sulfinamide,sulfonamide, or ester linkages or by amino, ether, or thioether linkagesdepending upon the nature of Y and X and the corresponding linkinggroups on Z¹ and the side chain. Preferably, when Y is —S, X is a bond.Where n is 2 or greater, each V may also be bound to each adjacent V bylinkages as described. Preferably, linkages are amides, esters orsulfonamides, most preferably amides. Accordingly, in some embodiments,each Y is —NH or —NR and each X is CO— or SO₂—.

In some embodiments, Z² is a spacer of formula —S_(A)—, —S_(B)—,—S_(A)—S_(B)— or —S_(B)—S_(A)—, wherein S_(A) and S_(B) are as definedbelow.

In some embodiments, Z² is selected from —S_(A)— or —S_(B)—S_(A)—, thatis, [side chain]-Z²Z¹ is [side chain]-S_(A)—Z¹ or [sidechain]-S_(B)—S_(A)—Z¹.

The Group S_(A)

S_(A) may be a single amino acid residue or a residue of an amino acidderivative, especially an amino acid derivative residue having asulfinyl or sulfonyl in place of the carboxy moiety at the C terminus.Additionally or alternatively, the single amino acid residue may have anoxygen or sulfur atom in place of the nitrogen atom at the N terminus.Preferably, S_(A) is a single amino acid residue.

In some embodiments, the amino acid may be selected from γ-Glu, α-Glu,α-Asp, β-Asp, Ala, β-Ala (3-aminopropanoic acid), and Gaba(4-aminobutanoic acid). It will be understood that amino acids may be Dor L, or a racemic or enantioenriched mixture. In some embodiments, theamino acid is an L-amino acid. In some embodiments, the amino acid is aD-amino acid.

In some preferred embodiments, S_(A) has a carboxylic acid substituent,with γ-Glu, α-Glu, α-Asp, and β-Asp, and sulfinyl and sulfonylderivatives thereof, being preferred. Accordingly, in some embodiments,the amino acid residue is:

where —X— is —CO—, —SO—, —SO₂—, preferably —CO—, and a is 1 or 2,preferably 2. In some embodiments, the carboxylic acid is an ester, andthe amino acid residue is:

where —X— is —CO—, —SO—, —SO₂—, preferably —CO—, and a is 1 or 2,preferably 2, and R is C₁₋₄alkyl or C₆aryl. Preferably R is C₁₋₄alkyl,preferably methyl or ethyl, more preferably ethyl.

Preferably, S_(A) is γ-Glu.

The Group S_(B)

S_(B) may be a linker of general formula:

wherein P_(U) is a polymeric unit and n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or10. One terminus of the linker S_(B) is an —NH, —NR, —S or —O—, whereinR may be alkyl, a protecting group or may form a linkage to another partof the polymeric unit; while the other is a bond or CO—, SO— or SO₂—.Accordingly, each polymeric unit P_(U) may be bound at each side byamide, sulfinamide, sulfonamide, or ester linkages or by amino, ether,or thioether linkages depending upon the nature of Y and X and thecorresponding linking groups on Z¹, S_(A), and Lys.

In some embodiments, each P_(U) may be independently a unit of formula:

wherein:

Y may be —NH, —NR, —S or —O, wherein R may be alkyl, a protecting groupor may form a linkage to another part of the spacer, with the remainingvalency forming a linkage to Z¹;

X may be a bond, CO—, SO—, or SO₂—, with the remaining valency forming alinkage to Lys;

and V is a bivalent organic moiety linking Y and X.

In some embodiments, V is the α-carbon of a natural or unnatural aminoacid, that is V is —CHR^(AA)—, wherein R^(AA) is an amino acid sidechain; or V is an optionally substituted C₁₋₆alkylene, or V is a chaincomprising one or more units of ethylene glycol in series, also known asPEG chain, for example, —CH₂CH₂(OCH₂CH₂)_(m)—O—(CH₂)_(p)—, where m is 0,1, 2, 3, 4, or 5, and p is 1, 2, 3, 4, or 5; when X is CO—, p ispreferably 1, 3, 4, or 5. Optional alkylene substituents include fluoro,methyl, hydroxy, hydroxymethyl, and amino.

Preferred P_(U) Units Include:

(i). Single amino acid residues: P_(U) ^(i);

(ii). Dipeptide residues: P_(U) ^(ii); and

(iii). Amino-(PEG)_(m)-carboxylic acid residues: P_(U) ^(iii),

and may be present in any combination or order. For example, S_(B) maycomprise one or more of each of P_(U) ^(i), P_(U) ^(ii), and P_(U)^(iii) in any order, or may comprise one or more units of P_(U) ^(i),P_(U) ^(ii), and P_(U) ^(iii) only, or one of more units selected fromP_(U) ^(i) and P_(U) ^(ii), P_(U) ^(i) and P_(U) ^(iii), or P_(U) ^(ii)and P_(U) ^(iii).

(i). P_(U) ^(i) Single Amino Acid Residues

Each P_(U) ^(i) may be independently selected from any natural orunnatural amino acid residue and, for example, may be selected from Gly,Pro, Ala, Val, Leu, Ile, Met, Cys, Phe, Tyr, Trp, His, Lys, Arg, Gln,Asn, α-Glu, γ-Glu, Asp, Ser Thr, Gaba, Aib, β-Ala, 5-aminopentanoyl,6-aminohexanoyl, 7-aminoheptanoyl, 8-aminooctanoyl, 9-aminononanoyl, and10-aminodecanoyl. Preferably, P_(U) ^(i) amino acid residues areselected from Gly, Ser, Ala, Thr, and Cys, more preferably from Gly andSer.

In some embodiments, S_(B) is —(P_(U) ^(i))_(n)—, wherein n is 1 to 8,more preferably 5 to 7, most preferably 6. In some preferredembodiments, S_(B) is —(P_(U) ^(i))_(n)—, n is 6 and each P_(U) ^(i) isindependently selected from Gly or Ser, with a preferred sequence being-Gly-Ser-Gly-Ser-Gly-Gly-.

(ii). P_(U) ^(ii) Dipeptide Residues

Each P_(U) ^(ii) may be independently selected from any dipeptideresidue comprising two natural or unnatural amino acid residues bound byan amide linkage. Preferred P_(U) ^(ii) dipeptide residues includeGly-Gly, Gly-Ser, Ser-Gly, Gly-Ala, Ala-Gly, and Ala-Ala, morepreferably Gly-Ser and Gly-Gly.

In some embodiments, S_(B) is —(P_(U) ^(ii))_(n)—, wherein n is 2 to 4,more preferably 3, and each P_(U) ^(ii) is independently selected fromGly-Ser and Gly-Gly. In some preferred embodiments S_(B) is —(P_(U)^(ii))_(n)—, n is 3 and each P_(U) ^(ii) is independently selected fromGly-Ser and Gly-Gly, with a preferred sequence being-(Gly-Ser)-(Gly-Ser)-(Gly-Gly).

Amino acids having stereogenic centres within P_(U) ^(i) and P_(U) ^(ii)may be racemic, enantioenriched, or enantiopure. In some embodiments,the or each amino acid is independently an L-amino acid. In someembodiments, the or each amino acid is independently a D-amino acid.

(iii). P_(U) ^(iii) Amino-(PEG)_(m)-Carboxylic Acid Residues

Each P_(U) ^(iii) may be independently a residue of general formula:

wherein m is 0, 1, 2, 3, 4, or 5, preferably 1 or 2, and p is 1, 3, 4,or 5, preferably 1.

In some embodiments, m is 1 and p is 1, that is, P_(U) ^(iii) is aresidue of 8-amino-3,6-dioxaoctanoic acid (also known as{2-[2-aminoethoxy]ethoxy}acetic acid and H₂N-PEG₃-COOH). This residue isreferred to herein as -PEG₃-.

In some embodiments, m is 2 and p is 1, that is, P_(U) ^(iii) is aresidue of 11-amino-3,6,9-trioxaundecanoic acid (also known asH₂N-PEG₄-COOH). This residue is referred to herein as -PEG₄-

In some embodiments, S_(B) is —(P_(U) ^(iii))_(n)—, wherein n is 1 to 3,more preferably 2.

In some preferred embodiments, S_(B) is selected from -PEG₃-PEG₃- and-PEG₄-PEG₄-.

Preferred —Z²—Z¹

It will be understood that the above preferences may be independentlycombined to give preferred —Z²—Z¹ combinations.

Some preferred —Z²—Z¹ combinations are shown below (in each case, ---indicates the point of attachment to the side chain of the amino acidcomponent of Ψ:

The presence of the polar group at the end of Z¹ is believed to enhancethe pharmacokinetic properties of the compound, for example, byincreasing half life and/or mean residence time, and reducing clearance.The linker may also contribute to these pharmacokinetic properties.Linkers comprising more than one amino acid unit (or moieties of similarsize) may improve pharmacokinetic properties compared to thoseconsisting of just one amino acid unit or the like. These properties mayenable the compound to be administered less frequently than anequivalent compound with the same peptide backbone but no modificationor a different modification (e.g. a substituent with an aliphatic fattychain lacking a polar group and/or having a shorter linker moiety).

Without wishing to be bound by any particular theory, the inventors havefound that, especially when longer linkers were included, the polar orcharged group at the end of Z¹ may be capable of participating in anundesirable intra-molecular interaction with the free N-terminus of themolecule which might compromise the beneficial effects of the polargroup on pharmacokinetics. The peptide backbones of the compoundsdescribed herein are believed to adopt relatively well-defined helicalsecondary structure, so the capacity of the polar group to engage insuch interactions may depend on its location within the molecule. Whenlocated towards the C-terminus, interaction with the N-terminus may berelatively unlikely. However, the inventors were surprised to find thatthe substituent could be located at residues 16 and 17 of the moleculewithout necessarily compromising the pharmacokinetic benefits obtained.

The term “conjugated” is used here to describe the physical attachmentof one identifiable chemical moiety to another, and the structuralrelationship between such moieties. It should not be taken to imply anyparticular method of synthesis.

The skilled reader will be well aware of suitable techniques that can beused to perform the coupling reactions using general syntheticmethodologies listed e.g. in “Comprehensive Organic Transformations, AGuide to Functional Group Preparations”, 2nd edition, Larock, R. C.;Wiley-VCH: New York, 1999. Such transformations may take place at anysuitable stage during the synthesis process.

Peptide Synthesis

The compounds of the present invention may be manufactured either bystandard synthetic methods, recombinant expression systems, or any otherstate of the art method. Thus the glucagon analogues may be synthesizedin a number of ways, including, for example, a method which comprises:

(a) synthesizing the peptide by means of solid-phase or liquid-phasemethodology, either stepwise or by fragment assembly, and isolation andpurifying of the final peptide product; or

(b) expressing a precursor peptide sequence from a nucleic acidconstruct that encodes the precursor peptide, recovering the expressionproduct, and modifying the precursor peptide to yield a compound of theinvention.

Expression is typically performed from a nucleic acid encoding theprecursor peptide, which may be performed in a cell or a cell-freeexpression system comprising such a nucleic acid.

It is preferred to synthesize the analogues of the invention by means ofsolid-phase or liquid-phase peptide synthesis. In this context,reference is made to WO 98/11125 and, among many others, Fields, G B etal., 2002, “Principles and practice of solid-phase peptide synthesis”.In: Synthetic Peptides (2nd Edition), and the Examples herein.

For recombinant expression, the nucleic acid fragments encoding theprecursor peptide will normally be inserted in suitable vectors to formcloning or expression vectors. The vectors can, depending on purpose andtype of application, be in the form of plasmids, phages, cosmids,mini-chromosomes, or virus, but also naked DNA which is only expressedtransiently in certain cells is an important vector. Preferred cloningand expression vectors (plasmid vectors) are capable of autonomousreplication, thereby enabling high copy-numbers for the purposes ofhigh-level expression or high-level replication for subsequent cloning.

In general outline, an expression vector comprises the followingfeatures in the 5′→3′ direction and in operable linkage: a promoter fordriving expression of the nucleic acid fragment, optionally a nucleicacid sequence encoding a leader peptide enabling secretion (to theextracellular phase or, where applicable, into the periplasma), thenucleic acid fragment encoding the precursor peptide, and optionally anucleic acid sequence encoding a terminator. They may compriseadditional features such as selectable markers and origins ofreplication. When operating with expression vectors in producer strainsor cell lines it may be preferred that the vector is capable ofintegrating into the host cell genome. The skilled person is veryfamiliar with suitable vectors and is able to design one according totheir specific requirements.

The vectors of the invention are used to transform host cells to producethe precursor peptide. Such transformed cells can be cultured cells orcell lines used for propagation of the nucleic acid fragments andvectors, and/or used for recombinant production of the precursorpeptides.

Preferred transformed cells are micro-organisms such as bacteria [suchas the species Escherichia (e.g. E. coli), Bacillus (e.g. Bacillussubtilis), Salmonella, or Mycobacterium (preferably non-pathogenic, e.g.M. bovis BCG), yeasts (e.g., Saccharomyces cerevisiae and Pichiapastoris), and protozoans. Alternatively, the transformed cells may bederived from a multicellular organism, i.e. it may be fungal cell, aninsect cell, an algal cell, a plant cell, or an animal cell such as amammalian cell. For the purposes of cloning and/or optimised expressionit is preferred that the transformed cell is capable of replicating thenucleic acid fragment of the invention. Cells expressing the nucleicfragment can be used for small-scale or large-scale preparation of thepeptides of the invention.

When producing the precursor peptide by means of transformed cells, itis convenient, although far from essential, that the expression productis secreted into the culture medium.

Efficacy

Binding of the relevant compounds to GLP-1 or glucagon (Glu) receptorsmay be used as an indication of agonist activity, but in general it ispreferred to use a biological assay which measures intracellularsignaling caused by binding of the compound to the relevant receptor.For example, activation of the glucagon receptor by a glucagon agonistwill stimulate cellular cyclic AMP (cAMP) formation. Similarly,activation of the GLP-1 receptor by a GLP-1 agonist will stimulatecellular cAMP formation. Thus, production of cAMP in suitable cellsexpressing one of these two receptors can be used to monitor therelevant receptor activity. Use of a suitable pair of cell types, eachexpressing one receptor but not the other, can hence be used todetermine agonist activity towards both types of receptor.

The skilled person will be aware of suitable assay formats, and examplesare provided below. The GLP-1 receptor and/or the glucagon receptor mayhave the sequence of the receptors as described in the examples. Forexample, the assays may employ the human glucagon receptor (Glucagon-R)having primary accession number GI:4503947 and/or the humanglucagon-like peptide 1 receptor (GLP-1R) having primary accessionnumber GI:166795283. (in that where sequences of precursor proteins arereferred to, it should of course be understood that assays may make useof the mature protein, lacking the signal sequence).

EC₅₀ values may be used as a numerical measure of agonist potency at agiven receptor. An EC₅₀ value is a measure of the concentration of acompound required to achieve half of that compound's maximal activity ina particular assay. Thus, for example, a compound having EC₅₀[GLP-1]lower than the EC₅₀[GLP-1] of glucagon in a particular assay may beconsidered to have higher GLP-1 receptor agonist potency than glucagon.

The compounds described in this specification are typically GluGLP-1dual agonists, as determined by the observation that they are capable ofstimulating cAMP formation at both the glucagon receptor and the GLP-1receptor. The stimulation of each receptor can be measured inindependent assays and afterwards compared to each other.

By comparing the EC₅₀ value for the GLP-1 receptor (EC₅₀[GLP-1-R]) withthe EC₅₀ value for the Glucagon receptor, (EC₅₀ [GlucagonR]) for a givencompound. the relative GLP-1R selectivity can be calculated as follows:Relative GLP-1R selectivity [compound]=(EC₅₀[GLP-1R])/(EC₅₀[Glucagon-R])

The term “EC₅₀” stands for the half maximal Effective Concentration,typically at a particular receptor, or on the level of a particularmarker for receptor function, and can refer to an inhibitory or anantagonistic activity, depending on the specific biochemical context.

Without wishing to be bound by any particular theory, a compound'srelative selectivity may allow its effect on the GLP-1 or glucagonreceptor to be compared directly to its effect on the other receptor.For example, the higher a compound's relative GLP-1 selectivity is, themore effective that compound may be on the GLP-1 receptor as compared tothe glucagon receptor. Typically the results are compared for glucagonand GLP-1 receptors from the same species, e.g. human glucagon and GLP-1receptors, or murine glucagon and GLP-1 receptors.

The compounds of the invention may have a higher relative GLP-1Rselectivity than human glucagon in that for a particular level ofglucagon-R agonist activity, the compound may display a higher level ofGLP-1R agonist activity (i.e. greater potency at the GLP-1 receptor)than glucagon. It will be understood that the absolute potency of aparticular compound at the glucagon and GLP-1 receptors may be higher,lower or approximately equal to that of native human glucagon, as longas the appropriate relative GLP-1R selectivity is achieved.

Nevertheless, the compounds of this invention may have a lower EC₅₀[GLP-1R] than human glucagon. The compounds may have a lowerEC₅₀[GLP-1-R] than glucagon while maintaining an EC₅₀ [Glucagon-R] thatis less than 10-fold higher than that of human glucagon, less than5-fold higher than that of human glucagon, or less than 2-fold higherthan that of human glucagon.

The compounds of the invention may have an EC₅₀ [Glucagon-R] that isless than two-fold that of human glucagon. The compounds may have anEC₅₀ [Glucagon-R] that is less than two-fold that of human glucagon andhave an EC₅₀ [GLP-1R] that is less than half that of human glucagon,less than a fifth of that of human glucagon, or less than a tenth ofthat of human glucagon.

The relative GLP-1R selectivity of the compounds may be between 0.05 and20. For example, the compounds may have a relative selectivity of0.05-0.20, 0.1-0.30, 0.2-0.5, 0.3-0.7, or 0.5-1.0; 1.0-2.0, 1.5-3.0,2.0-4.0 or 2.5-5.0; or 0.05-20, 0.075-15, 0.1-10, 0.15-5, 0.75-2.5 or0.9-1.1.

In certain embodiments, it may be desirable that EC₅₀ of any givencompound for both the Glucagon-R and GLP-1R, e.g. for the human glucagonand GLP-1 receptors, should be less than 1 nM.

Therapeutic Uses

The compounds of the invention may provide attractive treatment and/orprevention options for, inter alia, obesity and metabolic diseasesincluding diabetes, as discussed below.

Diabetes comprises a group of metabolic diseases characterized byhyperglycemia resulting from defects in insulin secretion, insulinaction, or both. Acute signs of diabetes include excessive urineproduction, resulting compensatory thirst and increased fluid intake,blurred vision, unexplained weight loss, lethargy, and changes in energymetabolism. The chronic hyperglycemia of diabetes is associated withlong-term damage, dysfunction, and failure of various organs, notablythe eyes, kidneys, nerves, heart and blood vessels. Diabetes isclassified into type 1 diabetes, type 2 diabetes and gestationaldiabetes on the basis on pathogenetic characteristics.

Type 1 diabetes accounts for 5-10% of all diabetes cases and is causedby auto-immune destruction of insulin-secreting pancreatic β-cells.

Type 2 diabetes accounts for 90-95% of diabetes cases and is a result ofa complex set of metabolic disorders. Type 2 diabetes is the consequenceof endogenous insulin production becoming insufficient to maintainplasma glucose levels below the diagnostic thresholds.

Gestational diabetes refers to any degree of glucose intoleranceidentified during pregnancy.

Pre-diabetes includes impaired fasting glucose and impaired glucosetolerance and refers to those states that occur when blood glucoselevels are elevated but below the levels that are established for theclinical diagnosis for diabetes.

A large proportion of people with type 2 diabetes and pre-diabetes areat increased risk of morbidity and mortality due to the high prevalenceof additional metabolic risk factors including abdominal obesity(excessive fat tissue around the abdominal internal organs), atherogenicdyslipidemia (blood fat disorders including high triglycerides, low HDLcholesterol and/or high LDL cholesterol, which foster plaque buildup inartery walls), elevated blood pressure (hypertension) a prothromboticstate (e.g. high fibrinogen or plasminogen activator inhibitor-1 in theblood), and proinflammatory state (e.g., elevated C-reactive protein inthe blood).

Conversely, obesity confers an increased risk of developingpre-diabetes, type 2 diabetes as well as e.g. certain types of cancer,obstructive sleep apnea and gall-bladder disease.

Dyslipidaemia is associated with increased risk of cardiovasculardisease. High Density Lipoprotein (HDL) is of clinical importance sincean inverse correlation exists between plasma HDL concentrations and riskof atherosclerotic disease. The majority of cholesterol stored inatherosclerotic plaques originates from LDL and hence elevatedconcentrations Low Density Lipoproteins (LDL) is closely associated withatherosclerosis. The HDL/LDL ratio is a clinical risk indictor foratherosclerosis and coronary atherosclerosis in particular.

Metabolic syndrome is characterized by a group of metabolic risk factorsin one person. They include abdominal obesity (excessive fat tissuearound the abdominal internal organs), atherogenic dyslipidemia (bloodfat disorders including high triglycerides, low HDL cholesterol and/orhigh LDL cholesterol, which foster plaque buildup in artery walls),elevated blood pressure (hypertension), insulin resistance and glucoseintolerance, prothrombotic state (e.g. high fibrinogen or plasminogenactivator inhibitor-1 in the blood), and proinflammatory state (e.g.,elevated C-reactive protein in the blood).

Individuals with the metabolic syndrome are at increased risk ofcoronary heart disease and other diseases related to othermanifestations of arteriosclerosis (e.g., stroke and peripheral vasculardisease). The dominant underlying risk factors for this syndrome appearto be abdominal obesity.

Without wishing to be bound by any particular theory, it is believedthat the compounds of the invention act as dual agonists both on thehuman glucagon-receptor and the human GLP1-receptor, abbreviated here asdual GluGLP-1 agonists. The dual agonist may combine the effect ofglucagon, e.g. on fat metabolism, with the effect of GLP-1, e.g. onblood glucose levels and food intake. They may therefore act toaccelerate elimination of excessive adipose tissue, induce sustainableweight loss, and improve glycemic control. Dual GluGLP-1 agonists mayalso act to reduce cardiovascular risk factors such as high cholesterol,high LDL-cholesterol or low HDL/LDL cholesterol ratios.

The compounds of the present invention can therefore be used in asubject in need thereof as pharmaceutical agents for preventing weightgain, promoting weight loss, reducing excess body weight or treatingobesity (e.g. by control of appetite, feeding, food intake, calorieintake, and/or energy expenditure), including morbid obesity, as well asassociated diseases and health conditions including but not limited toobesity linked inflammation, obesity linked gallbladder disease andobesity induced sleep apnea. The compounds of the invention may also beused for treatment of conditions caused by or associated with impairedglucose control, including metabolic syndrome, insulin resistance,glucose intolerance, pre-diabetes, increased fasting glucose, type 2diabetes, hypertension, atherosclerosis, arteriosclerosis, coronaryheart disease, peripheral artery disease and stroke, in a subject inneed thereof. Some of these conditions can be associated with obesity.However, the effects of the compounds of the invention on theseconditions may be mediated in whole or in part via an effect on bodyweight, or may be independent thereof.

The synergistic effect of dual GluGLP-1 agonists may also result inreduction of cardiovascular risk factors such as high cholesterol andLDL, which may be entirely independent of their effect on body weight.

Thus the invention provides the use of a compound of the invention inthe treatment of a condition as described above, in an individual inneed thereof.

The invention also provides a compound of the invention for use in amethod of medical treatment, particularly for use in a method oftreatment of a condition as described above.

In a preferred aspect, the compounds described may be used in treatingdiabetes, esp. type 2 diabetes.

In a specific embodiment, the present invention comprises use of acompound for treating diabetes, esp. type 2 diabetes in an individual inneed thereof.

In a not less preferred aspect, the compounds described may be used inpreventing weight gain or promoting weight loss.

In a specific embodiment, the present invention comprises use of acompound for preventing weight gain or promoting weight loss in anindividual in need thereof.

In a specific embodiment, the present invention comprises use of acompound in a method of treatment of a condition caused or characterisedby excess body weight, e.g. the treatment and/or prevention of obesity,morbid obesity, morbid obesity prior to surgery, obesity linkedinflammation, obesity linked gallbladder disease, obesity induced sleepapnea, prediabetes, diabetes, esp. type 2 diabetes, hypertension,atherogenic dyslipidimia, atherosclerosis, arteriosclerosis, coronaryheart disease, peripheral artery disease, stroke or microvasculardisease in an individual in need thereof.

In another aspect, the compounds described may be used in a method oflowering circulating LDL levels, and/or increasing HDL/LDL ratio.

In a specific embodiment, the present invention comprises use of acompound in a method of lowering circulating LDL levels, and/orincreasing HDL/LDL ratio in an individual in need thereof.

In another aspect, the compounds described may be used in a method oflowering circulating triglyceride levels.

Pharmaceutical Compositions

The compounds of the present invention may be formulated aspharmaceutical compositions prepared for storage or administration. Sucha composition typically comprises a therapeutically effective amount ofa compound of the invention, in the appropriate form, in apharmaceutically acceptable carrier.

The therapeutically effective amount of a compound of the presentinvention will depend on the route of administration, the type of mammalbeing treated, and the physical characteristics of the specific mammalunder consideration. These factors and their relationship to determiningthis amount are well known to skilled practitioners in the medical arts.This amount and the method of administration can be tailored to achieveoptimal efficacy, and may depend on such factors as weight, diet,concurrent medication and other factors, well known to those skilled inthe medical arts. The dosage sizes and dosing regimen most appropriatefor human use may be guided by the results obtained by the presentinvention, and may be confirmed in properly designed clinical trials.The compounds of the present invention may be particularly useful fortreatment of humans.

An effective dosage and treatment protocol may be determined byconventional means, starting with a low dose in laboratory animals andthen increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Numerous factors maybe taken into consideration by a clinician when determining an optimaldosage for a given subject. Such considerations are known to the skilledperson.

The term “pharmaceutically acceptable carrier” includes any of thestandard pharmaceutical carriers. Pharmaceutically acceptable carriersfor therapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remington's Pharmaceutical Sciences, MackPublishing Co. (A. R. Gennaro edit. 1985). For example, sterile salineand phosphate-buffered saline at slightly acidic or physiological pH maybe used. pH buffering agents may be phosphate, citrate, acetate,tris/hydroxymethyl)aminomethane (TRIS),N-Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS),ammonium bicarbonate, diethanolamine, histidine, which is a preferredbuffer, arginine, lysine, or acetate or mixtures thereof. The termfurther encompasses any agents listed in the US Pharmacopeia for use inanimals, including humans.

The term “pharmaceutically acceptable salt” refers to a salt of any oneof the compounds of the invention. Salts include pharmaceuticallyacceptable salts such as acid addition salts and basic salts. Examplesof acid addition salts include hydrochloride salts, citrate salts andacetate salts. Examples of basic salts include salts where the cation isselected from alkali metals, such as sodium and potassium, alkalineearth metals, such as calcium, and ammonium ions ⁺N(R³)₃(R⁴), where R³and R⁴ independently designates optionally substituted C₁₋₆-alkyl,optionally substituted C₂₋₆-alkenyl, optionally substituted aryl, oroptionally substituted heteroaryl. Other examples of pharmaceuticallyacceptable salts are described in “Remington's Pharmaceutical Sciences”,17th edition. Ed. Alfonso R. Gennaro (Ed.), Mark Publishing Company,Easton, Pa., U.S.A., 1985 and more recent editions, and in theEncyclopedia of Pharmaceutical Technology.

“Treatment” is an approach for obtaining beneficial or desired clinicalresults. For the purposes of this invention, beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. “Treatment” is an intervention performed with theintention of preventing the development or altering the pathology of adisorder. Accordingly, “treatment” refers to both therapeutic treatmentand prophylactic or preventative measures in certain embodiments. Thosein need of treatment include those already with the disorder as well asthose in which the disorder is to be prevented. By treatment is meantinhibiting or reducing an increase in pathology or symptoms (e.g. weightgain, hyperglycemia) when compared to the absence of treatment, and isnot necessarily meant to imply complete cessation of the relevantcondition.

The pharmaceutical compositions can be in unit dosage form. In suchform, the composition is divided into unit doses containing appropriatequantities of the active component. The unit dosage form can be apackaged preparation, the package containing discrete quantities of thepreparations, for example, packeted tablets, capsules, and powders invials or ampoules. The unit dosage form can also be a capsule, cachet,or tablet itself, or it can be the appropriate number of any of thesepackaged forms. It may be provided in single dose injectable form, forexample in the form of a pen. In certain embodiments, packaged formsinclude a label or insert with instructions for use. Compositions may beformulated for any suitable route and means of administration.Pharmaceutically acceptable carriers or diluents include those used informulations suitable for oral, rectal, nasal, topical (including buccaland sublingual), vaginal or parenteral (including subcutaneous,intramuscular, intravenous, intradermal, and transdermal)administration. The formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy.

Subcutaneous or transdermal modes of administration may be particularlysuitable for the compounds described herein.

Compositions of the invention may further be compounded in, or attachedto, for example through covalent, hydrophobic and electrostaticinteractions, a drug carrier, drug delivery system and advanced drugdelivery system in order to further enhance stability of the compound,increase bioavailability, increase solubility, decrease adverse effects,achieve chronotherapy well known to those skilled in the art, andincrease patient compliance or any combination thereof. Examples ofcarriers, drug delivery systems and advanced drug delivery systemsinclude, but are not limited to, polymers, for example cellulose andderivatives, polysaccharides, for example dextran and derivatives,starch and derivatives, poly(vinyl alcohol), acrylate and methacrylatepolymers, polylactic and polyglycolic acid and block co-polymersthereof, polyethylene glycols, carrier proteins, for example albumin,gels, for example, thermogelling systems, for example block co-polymericsystems well known to those skilled in the art, micelles, liposomes,microspheres, nanoparticulates, liquid crystals and dispersions thereof,L2 phase and dispersions there of, well known to those skilled in theart of phase behaviour in lipid-water systems, polymeric micelles,multiple emulsions, self-emulsifying, self-microemulsifying,cyclodextrins and derivatives thereof, and dendrimers.

Combination Therapy

A compound or composition of the invention may be administered as partof a combination therapy with an agent for treatment of obesity,hypertension, dyslipidemia or diabetes.

In such cases, the two active agents may be given together orseparately, and as part of the same pharmaceutical formulation or asseparate formulations.

Thus a compound or composition of the invention can further be used incombination with an anti-obesity agent, including but not limited to aglucagon-like peptide receptor 1 agonist, peptide YY or analoguethereof, cannabinoid receptor 1 antagonist, lipase inhibitor,melanocortin receptor 4 agonist, melanin concentrating hormone receptor1 antagonist, phentermine (alone or in combination with topiramate), acombination of norepinephrine/dopamine reuptake inhibitor and opioidreceptor antagonist (e.g. a combination of bupropion and naltrexone), ora serotonergic agent (e.g. lorcaserin).

A compound or composition of the invention can be used in combinationwith an anti-hypertension agent, including but not limited to anangiotensin-converting enzyme inhibitor, angiotensin II receptorblocker, diuretics, beta-blocker, or calcium channel blocker.

A compound or composition of the invention can be used in combinationwith a dyslipidaemia agent, including but not limited to a statin, afibrate, a niacin and/or a cholesterol absorption inhibitor.

Further, a compound or composition of the invention can be used incombination with an anti-diabetic agent, including but not limited to abiguanide (e.g. metformin), a sulfonylurea, a meglitinide or glinide(e.g. nateglinide), a DPP-IV inhibitor, an SGLT2 inhibitor, a glitazone,a different GLP-1 agonist, an insulin or an insulin analogue. In apreferred embodiment, the compound or salt thereof is used incombination with insulin or an insulin analogue, DPP-IV inhibitor,sulfonylurea or metformin, particularly sulfonylurea or metformin, forachieving adequate glycemic control. Examples of insulin analoguesinclude but are not limited to Lantus, Novorapid, Humalog, Novomix, andActraphane HM, Levemir and Apidra.

EXAMPLES Example 1: General Synthesis of Glucagon Analogues

Solid phase peptide synthesis (SPPS) was performed on a microwaveassisted synthesizer using standard Fmoc strategy in NMP on apolystyrene resin (TentaGel S Ram). HATU was used as coupling reagenttogether with DIPEA as base. Piperidine (20% in NMP) was used fordeprotection. Pseudoprolines: Fmoc-Phe-Thr(psiMe, Mepro)-OH andFmoc-Asp-Ser(psiMe, Mepro)-OH (purchased from NovaBiochem) were usedwhere applicable.

Abbreviations employed are as follows:

-   Boc: tert-butyloxycarbonyl-   ivDde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)3-methyl-butyl-   Dde: 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl-   DCM: dichloromethane-   DMF: N,N-dimethylformamide-   DIPEA: diisopropylethylamine-   EDT: 1,2-ethanedithiol-   EtOH: ethanol-   Et₂O: diethyl ether-   HATU:    N-[(dimethylamino)-1H-1,2,3-triazol[4,5-b]pyridine-1-ylmethylene]-N-methylmethanaminium    hexafluorophosphate N-oxide-   MeCN: acetonitrile-   NMP: N-methylpyrrolidone-   TFA: trifluoroacetic acid-   TIS: triisopropylsilane

Cleavage:

The crude peptide was cleaved from the resin by treatment with95/2.5/2.5% (v/v) TFA/TIS/water at room temperature (r.t.) for 2 hours.Most of the TFA was removed at reduced pressure and the crude peptidewas precipitated and washed with diethylether and allowed to dry toconstant weight at ambient temperature.

The following compounds were synthesised:

1 (SEQ ID NO: 69) H-H-Aib-QGTFTSDYSKYLDS-K([15-carboxy-pentadecanoyl]-isoGlu)-AAHDFVEWLLSA-NH2 2 (SEQ ID NO: 40)H-H-Aib-QGTFTSDYSKYLD-K([17-carboxy- heptadecanoyl]-isoGlu-Peg3-Peg3)-RAAKDFIEWLESA-NH2 3 (SEQ ID NO: 60)H-H-Aib-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2 4 (SEQ ID NO: 56)H-H-Ac4c-QGTFTSDYSKYLDE-K([17-carboxy- heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA-NH2 5 (SEQ ID NO: 41) H-H-Aib-QGTFTSDYSKYLD-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)- RAAKDFIEWLESA-NH2 6 (SEQ ID NO: 42)H-H-Aib-QGTFTSDYSKYLE-K([17-carboxy- heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH2 7 (SEQ ID NO: 43)H-H-Ac4c-QGTFTSDYSKYLD-K([17-carboxy- heptadecanoyl]-isoGlu-GSGSGG)-RAAKDFIEWLESA-NH2 8 (SEQ ID NO: 44) H-H-Aib-QGTFTSDYSKYLE-K([17-carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-  RAAHDFIEWLESA-NH2 9 (SEQ ID NO: 57)H-H-Ac4c-HGTFTSDYSKYLDE-K([17-carboxy- heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA-NH2 10 (SEQ ID NO: 55)H-H-Aib-QGTFTSDYSKYLDE-K([17-carboxy-heptadecanoyl]-isoGlu)-AAKDFIEWLESA-NH2 11 (SEQ ID NO: 58)H-H-Ac4c-QGTFTSDYSKYLDE-K([17-carboxy-heptadecanoyl]-isoGlu)-AAKDFIEWLESA-NH2 12 (SEQ ID NO: 59)H-H-Ac4c-QGTFTSDYSKYLDE-K([17-carboxy-heptadecanoyl]-isoGlu)-RAKDFIEWLESA-NH2 13 (SEQ ID NO: 61)H-H-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH2    14 (SEQ ID NO: 62)H-H-Ac4c-QGTFTSDYSKYLDERRAKDFI-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)- WLESA-NH2 15  (SEQ ID NO: 63)H-H-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)- A-NH2 16 (SEQ ID NO: 64)H-H-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLE-K([17-carboxy-heptadecanoyl]-isoGlu-GSGSGG)-  A-NH2

The acylated GLP-1 analogue semaglutide was also synthesised, and hasthe structure:H—H-[2-methyl-Ala]-EGTFTSDVSSYLEGQAA-K([17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3)-EFIAWLVRGRG-OH(SEQ ID NO: 70).

Example 2: Glucagon Receptor and GLP-1-Receptor Efficacy Assays

The cDNA encoding either the human glucagon receptor (Glucagon-R)(primary accession number P47871) or the human glucagon-like peptide 1receptor (GLP-1R) (primary accession number P43220) were synthesized andcloned into a mammalian expression vector containing a Zeocin resistancemarker.

The mammalian expression vectors encoding the Glucagon-R or the GLP-1-Rwere transfected into Chinese hamster ovary (CHO) cells by theAttractene method method. Stably expressing clones were obtained byZeocin selection (250 μg/mL) upon limited dilution of cells resistant tothe selection pressure. Glucagon-R and GLP-1-R cell clones expressingwere picked, propagated and tested in the Glucagon-R and GLP-1-Refficacy assays as described below. One Glucagon-R expressing clone andone GLP-1-R expressing clone were chosen for compound profiling.

CHO cells expressing the human Glucagon-R, or human GLP-1-R were seeded24 hours prior to the assay at 30,000 cells per well in 96-wellmicrotiter plates in culture in 100 μl growth medium. On the day ofanalysis, growth medium was removed and the cells were washed once with200 μl of assay buffer (Krebs-Ringer-buffer—KRBH). The buffer wasremoved and the cells were incubated for 15 min at room temperature in10 μl KRBH (KRBH+10 mM HEPES, 5 mM NaHCO3, 0.1% (V/V) BSA) with 0.1 mMIBMX in deionized water containing increasing concentrations of testpeptides. The reaction was stopped by the addition of lysis buffer (0.1%w/v BSA, 5 mM HEPES, 0.3% v/v Tween-20). After cell lysis for 10 min atroom temperature, lysates were transferred to 384-well plates and 10 μlof acceptor/donorbead mixture as contained in the AlphaScreen™ cAMPFunctional Assay Kit was added. After one hour of incubation at roomtemperature in the dark, the cAMP content was determined applying theAlphaScreen™ cAMP Functional Assay Kit from Perkin-Elmer according tomanufacturer instructions. EC₅₀ and relative efficacies compared toreference compounds (glucagon and GLP-1) were calculated applyingcomputer aided curve fitting. The GLP-1/glucagon ratio is calculated asdefined earlier. See Table 1.

TABLE 1 EC50 EC50 Ratio hGCGR hGLP-1R GLP-1/ Compound CHO-K1 [nM] CHO-K1[nM] Glucagon  1 0.21 nM 0.38 nM 1.81  2 0.13 nM 1.76 nM 13.54   3 1.48nM 0.70 nM 0.47  4 0.45 nM 0.70 nM 1.56  5 0.18 nM 0.83 nM 4.61  6 0.44nM 1.43 nM 3.25  7 0.11 nM 0.97 nM 8.82  8 0.31 nM 0.80 nM 2.58  9 0.07nM 0.97 nM 13.86  10 1.08 nM 0.41 nM 0.38 11 0.28 nM 0.56 nM 2.00 120.07 nM 0.48 nM 6.86 13 0.52 nM 0.33 nM 0.63 14 0.18 nM 0.60 nM 3.33 150.92 nM 0.61 nM 0.65 16 0.16 nM 0.53 nM 3.31

Example 3: Agonistic Activity on Endogenous GLP-1 Receptor

Agonistic activity of the test compounds on endogenous GLP-1 receptorswas determined using a murine insulinoma cell line. Intracellular cAMPwas used an indicator of receptor activation.

Cells were cultured for 24 h at a density of 10,000 cells/well in a384-well plate. Medium was removed and 10 μL KRBH buffer (NaCl 130 mM,KCl 3.6 mM, NaH₂PO₄ 0.5 mM, MgSO₄ 0.5 mM, CaCl₂ 1.5 mM) containing testcompound or GLP-1 (at increasing concentrations from 0.1 pM to 100 nM)or solvent control (0.1% (v/v) DMSO) was added to the wells for 15minutes at a temperature of 26° C.

The cellular cAMP content is measured using the AlphaScreen cAMPFunctional Assay Kit (Perkin Elmer). Measurement was performed using theEnvision (PerkinElmer) according to manufacturer's recommendations.

Results were converted into cAMP concentrations using a cAMP standardcurve prepared in KRBH buffer containing 0.1% (v/v) DMSO. The resultingcAMP curves were plotted as absolute cAMP concentrations (nM) over log(test compound concentration) and analyzed using the curve fittingprogram XLfit.

Parameters calculated to describe both the potency as well as theagonistic activity of each test compound on the endogenous GLP-1receptors were:

pEC50 (negative logarithmic value of EC50, a concentration resulting ina half-maximal elevation of cAMP levels, reflecting the potency of thetest compound);

Percent control (% CTL)(% cAMP elevation for each test compoundconcentration normalized based on the GLP-1-induced maximum cAMPresponse (100% CTL)). See Table 2.

TABLE 2 Compound EC50 [nM]  1 0.60 nM  2 0.69 nM  3 0.15 nM  4 0.40 nM 5 0.65 nM  6 0.54 nM  7 0.47 nM  8 0.36 nM  9 0.84 nM 10 0.60 nM 110.72 nM 12 0.81 nM 13 0.37 nM 14 0.38 nM 15 0.25 nM 16 0.34 nM

Example 4: Agonistic Activity on Endogenous Glucagon Receptor

Agonistic activity of the test compounds on endogenous glucagon receptorwas determined by measuring their effect on rate of glycogen synthesisin primary rat hepatocytes. Upon activation of the glucagon receptor, aninhibition of the glycogen synthesis rate is expected. Rate of glycogensynthesis was determined by counting the amount of radioactively labeledglucose incorporated into the cellular glycogen stores in a definedperiod of time.

Primary rat hepatocytes were cultured at a density of 40,000 cells/wellin a 24-well plate for 24 hours at 37° C. and 5% CO₂.

Medium was discarded and the cells washed with PBS. 180 μL of KRBH-basedbuffer containing 0.1% BSA and glucose at a concentration of 22.5 mM wasthen added to the wells, followed by test compound and 40 μCi/mlD-[U14C] glucose (20 μL each). Incubation was continued for 3 hours.

At the end of the incubation period, the incubation buffer was aspiratedand cells washed once with ice-cold PBS before lysis by incubation for30 min at room temperature with 100 μL 1 mol/l NaOH.

Cell lysates were transferred to 96-well filter plates and glycogenprecipitated by incubating the filter-plates for 120 min at 4° C.followed by washing the filter plates 4 times with ice-cold ethanol(70%). The resulting precipitates were filtered to dryness and theamount of incorporated ¹⁴C-glucose determined by using a Topcountscintillation counter according to manufacturer's recommendations.

Wells with vehicle controls (0.1% (v/v) DMSO in KRBH buffer) wereincluded as reference for non-inhibited glycogen synthesis (100% CTL).Wells without added D-[U¹⁴C] glucose were included as controls fornon-specific background signal (subtracted from all values). Endogenousglucagon peptide was used as a positive control.

All treatments were performed at least in duplicates.

Parameters calculated to describe both the potency as well as theagonistic activity of each test compound on the endogenous glucagonreceptor are pEC50 and % CTL.

% CTL is determined by calculating the percentage of CPM/well in thepresence of the test compound compared to the CPM/well of the vehiclecontrol after subtracting the background CPM/well:[CPM/well(basal)−CPM/well(sample)]*100/[CPM/well(basal)−CPM/well(control)]

An activator of the glucagon receptor will result in an inhibition ofthe glycogen synthesis rate and will give % CTL values between 0% CTL(complete inhibition) and 100% CTL (no observable inhibition).

The resulting activity curves were plotted as absolute counts (unit:cpm/sample) over log (test compound concentration) and analyzed usingthe curve fitting program XLfit.

pEC50 (negative logarithmic value of EC50) reflects the potency of thetest compound.

TABLE 3 Compound EC50 [nM]  1 0.85 nM  2 0.11 nM  3 0.94 nM  4 1.79 nM 5 0.21 nM  6 0.80 nM  7 0.34 nM  8 0.29 nM  9 0.11 nM 10 1.53 nM 110.95 nM 12 0.45 nM 13 0.43 nM 14 0.19 nM 15 3.63 nM 16 0.19 nM

The terms EC₅₀ and pEC₅₀ quoted in relation to GLP-1R activation couldequally be regarded as IC₅₀ and pIC₅₀ in relation to glycogen synthesis.

Example 5: Estimate of Pharmacokinetic Parameters

Pharmacokinetic parameters of the test compounds were determined afterintravenous administration to Han/Wistar rats. The acylated GLP-1analogue semaglutide was also tested for comparison purposes.

Male Wistar rats were obtained from Charles River (Germany) weighingapproximately 180 to 210 g at time of arrival at the test facility. Ratswere caged in European standard rat cages type IV with light cycle of12-hour dark and 12-hour light. During the study rats were housed instandard rat cages type III. Both diet Altromin 1324 (Altromin, Germany)and water was administered ad libitum during the whole experimentalperiod. The animals were housed in the test facility for at least 4 daysin order to assure proper acclimatization.

The compounds were first dissolved in 0.1% aqueous ammonia to a nominalconcentration of 2 mg/ml, and then diluted to the desired dosingstrength (10 μM) in sterile PBS containing 25 mM phosphate buffer, pH7.4. Intravenous injections corresponding to 20 nmol/kg were given via alateral tail vein.

Blood samples (200 μl) were collected from the periorbital plexus attime points 0.08, 0.25, 0.5, 1, 2, 4, 8, 24, 32 and 48 h post dosinginto K₃EDTA tubes and centrifuged for 5 minutes at 4° C. within 20minutes of sampling. Plasma samples (>100 μl) were transferred to96-well PCR plates, immediately frozen and kept at −20° C. untilanalysed for plasma concentration for the respective GLP-1-glucagoncompound using LC-MS/MS. Individual plasma concentration-time profileswere analysed by a non-compartmental approach using ToxKin™ version 3.2(Unilog IT Services), and the resulting pharmacokinetic parameters weredetermined. See Table 4.

TABLE 4 Mean Clearance Terminal Residence Compound (ml/min/kg) half life(h) Time (h) 2 0.11  9.1 13.6 3  0.056 23.4 28.7 4 0.11 13.7 17.6Semaglutide 0.10  9.0 11.4

The invention claimed is:
 1. A method of synthesizing a compound offormula (I):R¹—P¹—P²—R²  (I) wherein R¹ is H, C₁₋₄ alkyl, acetyl, formyl, benzoyl ortrifluoroacetyl; R² is OH or NH₂; P¹ is a peptide having the sequence:H—X2-X3-GTFTSDYSKYLDSΨAAHDFVEWLLSA wherein: X2 is selected from Aib,Ala, D-Ala, Ser, N-Me-Ser, Ac3c, Ac4c and Ac5c; and X3 is selected fromGln and His; P² is absent or is a sequence of 1-20 amino acid unitsindependently selected from the group consisting of Ala, Leu, Ser, Thr,Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr and Orn; or a pharmaceuticallyacceptable salt thereof; and Ψ is a residue of Lys, Arg, Orn or Cys inwhich the side chain is conjugated to a substituent having the formula—Z²—Z¹; —Z¹ is a fatty chain having a polar group at one end of thechain and a connection to Z², —X— at the end of the chain distal fromthe polar group, wherein the polar group comprises a carboxylic acid ora carboxylic acid bioisostere, a phosphonic acid, or a sulfonic acidgroup; and —X— is a bond, —CO—, —SO—, or —SO₂—; —Z²— is a spacer offormula:

wherein: each Y is independently —NH, —NR, —S or —O, where R is alkyl, aprotecting group or forms a linkage to another part of the spacer Z²;each X is independently a bond, CO—, SO—, or SO₂—; with the proviso thatwhen Y is —S, X is a bond; each V is independently a bivalent organicmoiety linking Y and X; and n is 1-10, said method comprising (i)providing an isolated precursor peptide and (ii) modifying (a) one ormore amino acids of the precursor peptide or (b) introducing to theprecursor peptide a substituent Z²—Z¹ to yield a compound of formula(I).
 2. The method of claim 1, wherein the method comprises expressing aprecursor peptide sequence from a nucleic acid construct that encodesthe precursor peptide, recovering the expression product, and modifyingthe precursor peptide to yield a compound of claim
 1. 3. The method ofclaim 2, comprising modifying the precursor peptide to introduce thesubstituent at residue Ψ.
 4. The method of claim 1, wherein P¹ has thesequence: H-Aib-QGTFTSDYSKYLDSΨAAHDFVEWLLSA.
 5. The method of claim 1,wherein the compound is: H—H-Aib-QGTFTSDYSKYLDSΨAAHDFVEWLLSA-NH₂.
 6. Themethod of claim 1, wherein —Z¹ is an acyl group of formula:A-B-Alk-(CO)— or a sulfonyl group of formula:A-B-Alk-(SO₂)—; wherein A is —COOH or a carboxylic acid bioisostere; Bis a bond, C₆arylene, or C₆arylene-O—; Alk is a saturated or unsaturatedfatty chain of 6 to 18 carbon atoms in length, optionally substitutedwith one or more substituents selected from fluoro, C₁₋₄alkyl,trifluoromethyl, hydroxymethyl, amino, hydroxyl, C₁₋₄alkoxy, oxo, andcarboxyl; —Z²— is —S_(A)—, —S_(A)—S_(B)—, or —S_(B)—S_(A)—; —S_(A)— is asingle amino acid residue selected from γ-Glu, α-Glu, α-Asp, β-Asp, Ala,β-Ala (3-aminopropanoic acid), and Gaba (4-aminobutanoic acid); —S_(B)—is a linker of general formula:

wherein n is 1-10 and each P_(U) is independently selected from P_(U)^(i) and P_(U) ^(iii); each P_(U) ^(i) is independently a natural orunnatural amino acid residue; and each P_(U) ^(iii) is independently aresidue of general formula:

wherein m is 0-5 and p is 1, 3, 4, or
 5. 7. The method of claim 1,wherein Z¹—Z² is selected from: (i)[17-Carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3; (ii)[17-Carboxy-heptadecanoyl]-isoGlu; (iii)[13-Carboxy-tridecanoyl]-isoGlu-Peg3-Peg3; (iv)[Carboxyphenoxynonanoyl]-isoGlu-Peg3-Peg3; (v)[13-Carboxy-tridecanoyl]-isoGlu-Peg4-Peg4; (vi)[17-Carboxy-heptadecanoyl]-Peg3-Peg3-isoGlu; (vii)[17-Carboxy-heptadecanoyl]-isoGlu-GSGSGG; and (viii)[17-Carboxy-heptadecanoyl]-AA-Peg3-Peg3.
 8. The method of claim 1,wherein P1 has the sequence:H-Aib-QGTFTSDYSKYLDS-K([15-carboxy-pentadecanoyl]-isoGlu)-AAHDFVEWLLSA.9. The method of claim 1, wherein the compound isH—H-Aib-QGTFTSDYSKYLDS-K([15-carboxy-pentadecanoyl]-isoGlu)-AAHDFVEWLLSA-NH₂.10. The method of claim 1, wherein Z²—Z¹ is[17-carboxy-heptadecanoyl]-isoGlu-Peg3-Peg3 or[17-carboxy-heptadecanoyl]-isoGlu-GSGSGG.